cloning DNA-binding proteins

taskan4 rey taskan4 at yahoo.es
Tue Jul 16 08:29:33 EST 2002


Thanks for your response.

> wouldn't you want to check the databases for TF
consensus 
> sequences in your
> promoter first? that could give you a clue about
what to look for and then
> see if they are there on a band-shift experiment.
> 

Yes, but not a single TF has been reported for this
particular fungus. How confident can I be if I find a
consensus sequence for a functionally-significant TF
from a related species? Are these binding sites well
conserved?

> yeast one hybrid experiments looking for TFs to a
promoter do not 
> generally
> work. i have only ever seen a handful of papers
describing such 
> experiments
> and in those cases, they were lucky enough not to
have too much background
> caused by spurious, low level interaction. MOST one
hybrid screens utilize
> short repetitive DNA sequence to isolate a single
unknown transcription
> factor which may bind to that site.

I'm actually thinking about trying to find significant
regions in the promoter first: internal or terminal
deletions and see what happens with expression. Or
linker scanning. But I'm sceptic about finding a short
important sequence this way. Am I right?


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